A brittle star is born: Ontogeny of luminous capabilities in Amphiura filiformis

Abstract

Bioluminescence is the production of visible light by living organisms thanks to a chemical reaction, implying the oxidation of a substrate called luciferin catalyzed by an enzyme, the luciferase. The luminous brittle star Amphiura filiformis depends on coelenterazine (i.e., the most widespread luciferin in marine ecosystems) and a luciferase homologous to the cnidarian Renilla luciferase to produce blue flashes in the arm's spine. Only a few studies have focused on the ontogenic apparitions of bioluminescence in marine organisms. Like most ophiuroids, A. filiformis displays planktonic ophiopluteus larvae for which the ability to produce light was not investigated. This study aims to document the apparition of the luminous capabilities of this species during its ontogenic development, from the egg to settlement. Through biochemical assays, pharmacological stimulation, and Renilla-like luciferase immunohistological detection across different developing stages, we pointed out the emergence of the luminous capabilities after the ophiopluteus larval metamorphosis into a juvenile. In conclusion, we demonstrated that the larval pelagic stage of A. filiformis is not bioluminescent compared to juveniles and adults.

Fig 1. Reproductive cycle of Amphiura filiformis at 14°C.

A: sexually mature adults (half-left male, half-right female), B: blastula, C: prism, D: early ophiopluteus, E: ophiopluteus, F: late ophiopluteus with larva rudiment, G: juvenile. The complete development of the brittle star Amphiura filiformis from the embryo to the juvenile takes around 30 days at 14°C. Scale bars represent (A), 500 μm, (B-C), 30 μm, (D), 40 μm, (E-F) 50 μm, (G), 80 μm. The color gradation from green (presence of coelenterazine) to blue (emergence of luminescence) on the arrow represents the evolution of luminous capabilities.

Fig 2. Luminescence capabilities of Amphiura filiformis larval stages from male and female gonads to 32 days post-fertilization (dpf).

(A) Coelenterazine content (ng g^-1) is significantly higher in the female gonads, (B) Luciferase activity (10⁹ q g^-1 s^-1) raised at 32 dpf, (C) Total light emission (10⁹ q g^-1) with KCl stimulation (10⁹ q g^-1) increased significantly 32 days after fertilization. Values are expressed as mean ± s.e.m. Asterisks indicate statistical differences between each larval stage with n≧6 (i) Kruskal–Wallis ANOVA and Wilcoxon multiple comparisons for coelenterazine content and total light emission, (ii) ANOVA and Tukey’s test for the luciferase activity; *P<0.05.

Fig 3. Immunolocalization of the Renilla-like luciferase (in red) in Amphiura filiformis larval stages; (A) 15 days, (B) 25 days, (C) 32 days post-fertilization, and (D) juveniles.

aa, arm article; di, disk; dt, digestive tract; es, oesophagus; jr, juvenile rudiment; jw, jaws; mo, mouth opening; po, podia; pd, pentameric development; sk, skeleton rod; sp, spine; td, tip of the developing arm. Scale bar: 40 μm.